The role of Eomesodermin (Eomes), a T-box transcription factor, in cardiac development is incompletely understood. Eomes was first described by the PI and colleagues, and was shown to be required both in Xenopus and in mouse development. Eomes responds to TGF-a signaling in an immediate-early fashion, through a 571 bp enhancer element (ARE) involving Smad/FAST proteins. Shown herein, Eomes is required in heart development. Our hypothesis consists of a four-step mechanism. 1) TGF-a signaling results in cooperation of Eomes with Smad proteins in trans-activating downstream target genes. 2) Eomes-Smad complexes bind to sites within the 571 bp Eomes ARE, maintaining Eomes expression. 3) Proper Eomes dosage is required for downstream gene activations. 4) Sustained Eomes expression is required in ventricular cardiomyocytes and bulbus cordis. The proposed research will further elucidate this essential cardiac Eomes pathway. AIM 1. A dominant-negative (DN) activin receptor prevents Eomes activation of mesodermal genes. Wild type and DN-Smads are used to determine if Eomes interacts with Smads in activating target genes. The Eomes promoter driving a human globin reporter is used to ask if Eomes interacts functionally with Smads. AIM 2. Eomes binding sites lie in close proximity to Smad sites in a functionally important region of the ARE. Promoter mutation, gel shift, and a promoter bioassay are used to characterize the regions within the Eomes ARE that regulate Eomes-Smad signaling, and identify their interacting proteins. AIM 3. A conditional Eomes isoform is used to examine if the dose of Eomes is important in normal cardiac development. AIM 4. In situ hybridization is used to determine the full range of cardiac cell types expressing Eomes; a conditional dominant-negative isoform of Eomes examines the requirement for Eomes in the heart.